Photoinactivation of Escherichia coli using porphyrin derivatives with different number of cationic charges

The photodynamic effect of meso-substituted cationic porphyrins, 5-[4-(trimethylammonium)phenyl]-10,15,20-tris(2,4,6-trimethoxyphenyl)porphyrin iodide 1, 5,10-di(4-methylphenyl)-15,20-di(4-trimethylammoniumphenyl)porphyrin iodide 2 and 5-(4-trifluorophenyl)-10,15,20-tris(4-trimethylammoniumphenyl)porphyrin iodide 3, have been investigated in both homogeneous medium bearing photooxidizable substrates and in vitro on a typical gram-negative bacterium Escherichia coli. Absorption and fluorescence spectroscopic studies were compared in N,N-dimethylformamide. Fluorescence quantum yields (varphiF) of 0.10, 0.06 and 0.08 were calculated for porphyrins 1, 2 and 3, respectively. The singlet molecular oxygen, O2(1Deltag), production was evaluated using 9,10-dimethylanthracene yielding values of 0.66, 0.36 and 0.42 for porphyrins 1, 2 and 3, respectively. Guanosine 5'-monophosphate was used as biological substrate model. Similar decomposition of guanosine 5'-monophosphate was obtained using these cationic porphyrins as sensitizer. In biological medium, photosensitized inactivation of E. coli was analyzed using cells without and with one washing step. E. coli cultures were treated with sensitizer at 37 degrees C for 30 min in dark. In both procedures, a higher photoinactivation of cells (>99.999%) was found for cells treated with 10 microM of tricationic porphyrin 3 and irradiated for 5 min with visible light. Porphyrins 1 and 2 only show an important photodamage when the cells are irradiated without washing step. These results indicated that the tetracationic porphyrin 3 could be a promising sensitizer with potential applications in the photoinactivation of bacterial cells by photodynamic therapy.     

Photodynamic inactivation of recombinant bioluminescent Escherichia coli by cationic porphyrins under artificial and solar irradiation

A faster and simpler method to monitor the photoinactivation process of Escherichia coli involving the use of recombinant bioluminescent bacteria is described here. Escherichia coli cells were transformed with luxCDABE genes from the marine bioluminescent bacterium Vibrio fischeri and the recombinant bioluminescent indicator strain was used to assess, in real time, the effect of three cationic meso-substituted porphyrin derivatives on their metabolic activity, under artificial (40 W m⁻²) and solar irradiation (≈620 W m⁻²). The photoinactivation of bioluminescent E. coli is effective (>4 log bioluminescence decrease) with the three porphyrins used, the tricationic porphyrin Tri-Py⁺-Me-PF being the most efficient compound. The photoinactivation process is efficient both with solar and artificial light, for the three porphyrins tested. The results show that bioluminescence analysis is an efficient and sensitive approach being, in addition, more affordable, faster, cheaper and much less laborious than conventional methods. This approach can be used as a screening method for bacterial photoinactivation studies in vitro and also for the monitoring of the efficiency of novel photosensitizer molecules. As far as we know, this is the first study involving the use of bioluminescent bacteria to monitor the antibacterial activity of porphyrins under environmental conditions.     

Sewage bacteriophage photoinactivation by cationic porphyrins: a study of charge effect.

Photodynamic therapy has been used to inactivate microorganisms through the use of targeted photosensitizers. Recently the inactivation of bacteria in residual waters has been reported, but nothing is known about photoinactivation of environmental bacteriophages, which are often used as indicators of human enteric viruses. In this study we tested the effect of six cationic porphyrin derivatives with two to four charges on the photoinactivation of a sewage bacteriophage. A phage suspension of 5 x 10(7) PFU mL(-1) was exposed to white light (40 W m(-2)), during 270 min, at three photosensitizer concentrations (0.5, 1.0 and 5.0 microM). Tetra- and tricationic porphyrins inactivated the T4-like sewage phage to the limits of detection, but dicationic porphyrins did not lead to a significant decrease in phage viability. At the highest photosensitizer concentration (5.0 microM), the phage was completely inactivated (>99.9999% of inactivation, reduction of 7.2 log) after 270 min by the tetracationic porphyrin. Two of the tricationic derivatives also led to phage inactivation to the limit of detection. The rate of bacteriophage photoinactivation and the efficiency of the photosensitizer appeared to vary with the charge and with the substituents in the meso-positions of the porphyrin macrocycle. Tetra- and tricationic porphyrins can, therefore, be used as a new method for inactivating sewage bacteriophages that are frequently used as human enteric virus indicators. The complete inactivation of viruses with low light intensity means that this methodology can be used even on cloudy days and during winter, opening the possibility to develop new technologies for wastewater treatment.     

Interaction of cationic <Emphasis Type="Italic">meso</Emphasis>-porphyrins with liposomes,mitochondria and erythrocytes

Two series of cationic porphyrins meso-(3N-methylpyridinium)phenylporphyrin (3P1, 3P2c, 3P2t, 3P3 and 3P4) and meso-(4N-methylpyridinium)phenylporphyrin (4P1, 4P2c, 4P2t, 4P3 and 4P4) were studied to obtain a comprehensive understanding of factors that influence the binding of cationic porphyrins to liposomes and mitochondria, as well as their photodynamic efficiencies in erythrocytes. Binding and photodynamic efficiency were found to be inversely proportional to the number of positively charged groups and directly proportional to n-octanol/water partition coefficients (log P_OW), except for the cis molecules 3P2c and 4P2c. In the cis molecules, binding and photodynamic efficiency were much higher than expected, indicating that specific interactions not accounted by log P_OW enhance photodynamic efficiency. The effect of mitochondrial transmembrane electrochemical potentials on cationic porphyrin binding constants was estimated to be as large as 15%, and may be useful to selectively target this organelle when promoting photodynamic therapy to induce apoptosis.     

Photodynamic effect of meso-(aryl)porphyrins and meso-(1-methyl-4-pyridinium)porphyrins on HaCaT keratinocytes

Sixteen porphyrins, including neutral, anionic and cationic meso-(aryl)porphyrins and meso-(1-methyl-4-pyridinium)porphyrins were herein evaluated in terms of their photosensitizing properties against HaCaT keratinocytes. After an initial screening, the cationic porphyrins were studied in more details, by both determining their log P_OW and performing PDT assays in lower porphyrin concentrations. Porphyrins presenting two or more adjacent positively charged groups, directly linked to the macrocycle meso positions, appeared to be the most effective photosensitizers. The present study also included the dicationic 5,10-diphenyl-15,20-di(1-methylpyridinium-4-yl)porphyrin (14b), which has previously shown promising results on a psoriasis-like in vivo model. Overall results indicated that the beneficial effect related to porphyrins on psoriasis can be related to the decreasing of keratinocyte viability. Furthermore, some of the cationic porphyrins studied appeared as candidates to be utilized as photosensitizers for psoriasis treatment.     

Photodynamic inactivation of Escherichia coli by novel meso-substituted porphyrins by 4-(3-N,N,N-trimethylammoniumpropoxy)phenyl and 4-(trifluoromethyl)phenyl groups.

The photodynamic effect of novel cationic porphyrins, with different pattern of meso-substitution by 4-(3-N,N,N-trimethylammoniumpropoxy)phenyl (A) and 4-(trifluoromethyl)phenyl (B) groups, have been studied in both solution bearing photooxidizable substrates and in vitro on a typical Gram-negative bacterium Escherichia coli. In these sensitizers, the cationic groups are separated from the macrocycle ring by a propoxy spacer. Thus, the charges have a high mobility and a minimal influence on photophysical properties of the porphyrin. These compounds produce singlet molecular oxygen, O2(1Delta(g)), with quantum yields of approximately 0.41-0.53 in N,N-dimethylformamide. In methanol, the l-tryptophan photodecomposition increases with the number of cationic charges in the sensitizer. In vitro investigations show that cationic porphyrins are rapidly bound to E. coli cells in approximately 5 min. A higher binding was found for A3B3+ porphyrin, which is tightly bound to cells still after three washing steps. Photosensitized inactivation of E. coli cellular suspensions follows the order: A3B3+ > A44+> ABAB2+ > AB3+. Under these conditions, a negligible effect was found for 5,10,15,20-tetra(4-sulfonatophenyl)porphyrin (TPPS4(4-)) that characterizes an anionic sensitizer. Also, the results obtained for these new cationic porphyrins were compared with those of 5,10,15,20-tetra(4-N,N,N-trimethylammonium phenyl)porphyrin (TTAP4+), which is a standard active sensitizer established to eradicate E. coli. The photodynamic activity of TTAP4+ is quite similar to that produced by A4(4+). Studies in an anoxic condition indicate that oxygen is necessary for the mechanism of action of photodynamic inactivation of bacteria. The higher photodynamic activity of A3B3+ was confirmed by growth delay experiments. Photodynamic inactivation capacities of these sensitizers were also evaluated in E. coli cells immobilized on agar surfaces. Under these conditions, A3B3+ porphyrin retains a high activity to inactivate localized bacterial cells. Therefore, tricationic porphyrin A3B3+ is an interesting sensitizer with potential applications in photodynamic inactivation of bacteria in liquid suspensions or on surfaces.     

Identification of genes involved in the mutualistic colonization of the nematode <Emphasis Type="Italic">Heterorhabditis bacteriophora</Emphasis> by the bacterium <Emphasis Type="Italic">Photorhabdus luminescens</Emphasis>

Background  

Photorhabdus are Gram negative entomopathogenic bacteria that also have a mutualistic association with nematodes from the family Heterorhabditis. An essential part of this symbiosis is the ability of the bacterium to colonize the gut of the freeliving form of the nematode called the infective juvenile (IJ). Although the colonization process (also called transmission) has been described phenomonologically very little is known about the underlying molecular mechanisms. Therefore, in this study, we were interested in identifying genes in Photorhabdus that are important for IJ colonization.     

Cationic Ester Porphyrins Cause High Levels of Phototoxicity in Tumor Cells and Induction of Apoptosis in HeLa Cells

A series of cationic ester porphyrins are much more cytotoxic to tumor cells than photofrin, meso‐tetrakis(1‐methylpyridinium‐4‐yl)porphyrin (TMPyP4), and cisplatin. The lowest IC₅₀ value for SGC7901 is ca. 6 nM in vitro with irradiation. These porphyrins also exhibited the most potent photo‐induced cytotoxicity without photobleaching. HeLa Cell apoptosis induced by cationic ester porphyrins after illumination was examined by flow cytometric analysis, staining assays with propidium iodide and annexin V FITC‐PI, and further confirmed by observing morphological changes in the cells. The result of this study indicates that these cationic ester porphyrins may be applied in photodynamic therapy (PDT) in the future.     

Comparison of the in vitro and in vivo susceptibilities of Burkholderia mallei to Ceftazidime and Levofloxacin

Background  

Burkholderia mallei is a zoonotic Gram negative bacterium which primarily infects solipeds but can cause lethal disease in humans if left untreated. The effect of two antibiotics with different modes of action on Burkholderia mallei strain ATCC23344 was investigated by using in vitro and in vivo studies.     

Binding of Porphyrins by the Tumor-Specific Lectin, Jacalin [Jack Fruit (Artocarpus integrifolia) Agglutinin]

Jacalin (Artocarpus integrifolia agglutinin) specifically recognizes thetumor-associated T-antigenic disaccharide structure,Gal13GalNAc. Porphyrins and their derivatives are currently used asphotosensitizers in photodynamic therapy to treat malignant tumors. In thisstudy, the interaction of several free base porphyrins and their metalderivatives with jacalin is investigated by absorption and fluorescencespectroscopy. Each lectin subunit was found to bind one porphyrin moleculeand the association constants were estimated to be in the range of2.4×10³M^–1 to 1.3×10⁵M^–1 at room temperaturefor the interaction of different porphyrins with jacalin. These values arein the same range as those obtained for the interaction of monosaccharidesto jacalin. Both free lectin and lectin saturated with the specificsaccharide were found to bind different porphyrins with comparable bindingstrength indicating that porphyrin binding takes place at a site differentfrom the sugar binding site. Further, both anionic and cationic porphyrinswere found to interact with the lectin with comparable affinity, clearlyindicating that the charge on the porphyrin does not play any role in thebinding process and that most likely the interaction is mediated byhydrophobic forces. These results suggest that jacalin and other lectins maypotentially be useful for targeted delivery of porphyrins to tumor tissuesin photodynamic therapy.     

Simultaneous differential detection of Chlamydophila abortus, Chlamydophila pecorum and Coxiella burnetii from aborted ruminant's clinical samples using multiplex PCR

Background  

Chlamydiosis and Q fever, two zoonosis, are important causes of ruminants' abortion around the world. They are caused respectively by strictly intracellular and Gram negative bacterium Chlamydophila abortus (Cp. abortus) and Coxiella burnetii (C. burnetii). Chlamydophila pecorum (Cp. pecorum) is commonly isolated from the digestive tract of clinically inconspicuous ruminants but the abortive and zoonotic impact of this bacterium is still unknown because Cp. pecorum is rarely suspected in abortion cases of small ruminants. We have developed a multiplex PCR (m-PCR) for rapid simultaneous differential detection of Cp. abortus, Cp. pecorum and C. burnetii in clinical samples taken from infected animals.     

Photodynamic antimicrobial activity of new porphyrin derivatives against methicillin resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Methicillin resistant Staphylococcus aureus (MRSA) with multiple drug resistance patterns is frequently isolated from skin and soft tissue infections that are involved in chronic wounds. Today, difficulties in the treatment of MRSA associated infections have led to the development of alternative approaches such as antimicrobial photodynamic therapy. This study aimed to investigate photoinactivation with cationic porphyrin derivative compounds against MRSA in in-vitro conditions. In the study, MRSA clinical isolates with different antibiotic resistance profiles were used. The newly synthesized cationic porphyrin derivatives (P_M, P_E, P_PN, and P_PL) were used as photosensitizer, and 655 nm diode laser was used as light source. Photoinactivation experiments were performed by optimizing energy doses and photosensitizer concentrations. In photoinactivation experiments with different energy densities and photosensitizer concentrations, more than 99% reduction was achieved in bacterial cell viability. No decrease in bacterial survival was observed in control groups. It was determined that there was an increase in photoinactivation efficiency by increasing the energy dose. At the energy dose of 150 J/cm² a survival reduction of over 6.33 log₁₀ was observed in each photosensitizer type. While 200 μM P_M concentration was required for this photoinactivation, 12.50 μM was sufficient for P_E, P_PN, and P_PL. In our study, antimicrobial photodynamic therapy performed with cationic porphyrin derivatives was found to have potent antimicrobial efficacy against multidrug resistant S. aureus which is frequently isolated from wound infections.     

The photodynamic effect: the comparison of chemiexcitation by luminol and phthalhydrazide

The presence of light, oxygen and photosensitizer (organic dye) is required for the photodynamic effect. Light and photosensitizer are harmless by themselves, but when combined with oxygen, reactive oxygen species (ROS) can be produced. This photodynamic effect is used in photodynamic therapy (PDT); the production of ROS as lethal cytotoxic agents can inactivate tumor cells. However, during PDT, there are many difficulties, so it is not possible to excite the photosensitizer using a laser, a source of light at the wavelengths specific to the photosensitizer (in visible region of the spectrum). Chemiluminescence is the light emission as a result of a chemical reaction. It is possible to use a chemiluminescent mixture to excite the photosensitizer even if the light emission does not conform to the absorption maximum of the photosensitizer. Luciferin and luminol have been used as chemiluminescent compounds (energizers) for the excitation of the photosensitizers. The aim of this work was to compare the chemiexcitation of some selected photosensitizers (e.g. fluorescein, eosin, methylene blue, hypericin and phthalocyanines) by chemiluminescent mixtures containing luminol (high chemiluminescent quantum yield) or phthalhydrazide (low chemiluminescent quantum yield) on some Gram‐positive (Enterococcus faecalis, Staphylococcus aureus) and Gram‐negative (Pseudomonas aeruginosa, E. coli) bacteria and some cell lines (NIH3T3 and MCF7). The efficiency of the chemiexcitation was dependent on the kind of the photosensitizer and on the type of the bacterial strain or cell line and was independent of the energizers. Copyright © 2010 John Wiley & Sons, Ltd.     

In vitro antimicrobial activity of light-activated phthalocyanines

Background

Photodynamic antimicrobial therapy (PACT) is proposed as a topical, non-invasive approach suitable for treatment of locally occurring infection. Research of photosensitizers, (PS) as well as their development, is aimed at finding effective antimicrobial substances which would have a broad-spectrum potency. The aim of this paper is to evaluate the antimicrobial effect of phthalocyanine (Pc) derivatives.

Methods

Fifteen different Pc compounds were investigated. Their photokilling activity was tested on Staphylococcus aureus, Escherichia coli and Candida albicans. After treating of microbial cells with Pc at the concentrations: 1 mg/l, 2 mg/l, 4 mg/l, 8 mg/l for 30 minutes, the cultures were irradiated with low-power laser light at a wavelength of 670 nm (20 J/cm², 40 J/cm²). The effectiveness of photoinactivation was evaluated based on the decrease in number (log₁₀) of viable bacteria.

Results

Eight Pc compounds tested showed antibacterial effects against S. aureus, but only four were effective against E. coli and two against C. albicans. The most effective photosensitizers were amphiphilic sulphonated zinc Pc compounds [(3-diethylammonium)-propylsulphonamide citrate (Pc3) and cationic tetramethylenepyridinium chloride of hydroxyaluminum Pc (Pc7)].

Conclusions

The most efficient phthalocyanines (Pc3, Pc7) cause a significant decrease in viable counts of all tested microbes.     

The core genome of the anaerobic oral pathogenic bacterium <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis>

Background  

The Gram negative anaerobic bacterium Porphyromonas gingivalis has long been recognized as a causative agent of periodontitis. Periodontitis is a chronic infectious disease of the tooth supporting tissues eventually leading to tooth-loss. Capsular polysaccharide (CPS) of P. gingivalis has been shown to be an important virulence determinant. Seven capsular serotypes have been described. Here, we used micro-array based comparative genomic hybridization analysis (CGH) to analyze a representative of each of the capsular serotypes and a non-encapsulated strain against the highly virulent and sequenced W83 strain. We defined absent calls using Arabidopsis thaliana negative control probes, with the aim to distinguish between aberrations due to mutations and gene gain/loss.     

Electrostatic binding of substituted metal phthalocyanines to enterobacterial cells: Its role in photodynamic inactivation

The effect of ionic substituents in zinc and aluminum phthalocyanine molecules and of membrane surface charge on the interaction of dyes with artificial membranes and enterobacterial cells, as well as on photosensitization efficiency was studied. It has been shown that increasing the number of positively charged substituents enhances the extent of phthalocyanine binding to Escherichia coli cells. This, along with the high quantum yield of singlet oxygen generation, determines efficient photodynamic inactivation of Gram-negative bacteria by zinc and aluminum octacationic phthalocyanines. The effect of Ca²⁺ and Mg²⁺ cations and pH on photodynamic inactivation of enterobacteria in the presence of octacationic zinc phthalocyanine has been studied. It has been shown that effects resulting in lowering negative charge on outer membrane protect bacteria against photoinactivation, which confirms the crucial role in this process of the electrostatic interaction of the photosensitizer with the cell wall. Electrostatic nature of binding is consistent with mainly electrostatic character of dye interactions with artificial membranes of different composition. Lower sensitivity of Proteus mirabilis to photodynamic inactivation, compared to that of E. coli and Salmonella enteritidis, due to low affinity of the cationic dye to the cells of this species, was found.     

Real‐time imaging of photodynamic action in bacteria

Fluorescence imaging studies of the processes leading to photodynamic inactivation of bacteria have been limited due to the small size of microorganisms as well as by the faint fluorescence of most photosensitizers. A versatile method based on highly‐sensitive fluorescence microscopy is presented which allows to study, in real time, the incorporation of photosensitizers inside S. aureus upon photodynamic action. The method takes advantage of the fluorescence enhancement of phenothiazine and porphyrin photosensitizers upon entering the bacterial cytosol after the cell wall has been compromised. In combination with typical assays, such as the addition of specific enhancers of reactive oxygen species, it is possible to extract mechanistic information about the pathway of photodynamic damage at the single‐cell level. Imaging experiments in deuterated buffer strongly support a Type‐I mechanism for methylene blue and a very minor role of singlet oxygen.

     

Mechanistic Insight into the Photodynamic Effect Mediated by Neutral Red and a New Azine Compound in Staphylococcus aureus Cells

The photodynamic activity of Neutral Red and the new monobrominated Neutral Red was studied in suspensions of Staphylococcus aureus. The effect of mannitol and sodium azide in the presence of 25 μm photosensitizer on lethal photosensitization were investigated. The results of the mechanistic evaluation of Neutral Red showed that both mannitol and sodium azide produced a completed protective effect after irradiation without significant differences between them. The evaluation of monobrominated Neutral Red also showed a protective effect of microorganisms with the addition of mannitol. Although sodium azide produced a protective effect of the photoinactivation, it was incomplete and less than that exhibited by mannitol. The results indicate that the starting reagent, Neutral Red, is a producer of radical species, acting through a type I mechanism, whereas the halogenated derivative of Neutral Red produced reactive oxygen species and a contribution of singlet molecular oxygen cannot be discarded in the photoinactivation of Staphylococcus aureus cells. These results, analyzed together with the previously evaluated properties of the dyes, allow us to explain the differences observed in the photoinactivation of Staphylococcus aureus mediated by both azine photosensitizers.     

Superoxide dismutase is upregulated in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> following protoporphyrin-mediated photodynamic inactivation and does not directly influence the response to photodynamic treatment

Background  

Staphylococcus aureus, a major human pathogen causes a wide range of disease syndromes. The most dangerous are methicillin-resistant S. aureus (MRSA) strains, resistant not only to all β-lactam antibiotics but also to other antimicrobials. An alarming increase in antibiotic resistance spreading among pathogenic bacteria inclines to search for alternative therapeutic options, for which resistance can not be developed easily. Among others, photodynamic inactivation (PDI) of S. aureus is a promising option. Photodynamic inactivation is based on a concept that a non toxic chemical, called a photosensitizer upon excitation with light of an appropriate wavelength is activated. As a consequence singlet oxygen and other reactive oxygen species (e.g. superoxide anion) are produced, which are responsible for the cytotoxic effect towards bacterial cells. As strain-dependence in photodynamic inactivation of S. aureus was observed, determination of the molecular marker(s) underlying the mechanism of the bacterial response to PDI treatment would be of great clinical importance. We examined the role of superoxide dismutases (Sod) in photodynamic inactivation of S. aureus as enzymes responsible for oxidative stress resistance.     

Unbiased random mutagenesis contributes to a better understanding of the virulent behaviour of Paenibacillus larvae

Aims

American foulbrood, caused by the Gram‐positive bacteria Paenibacillus larvae, is one of the most severe bacterial diseases of the European honey bee. The bacterium has been known for long, but only the last decade the mechanisms used by the pathogen to cause disease in its host are starting to unravel. In this study, the knowledge of this virulent behaviour is expanded and several possible virulence factors are suggested.

Methods and Results

Identification of possible virulence factors has been done by random mutagenesis to ensure an unbiased approach. A library of mutants was tested for a significant difference in virulence using in vitro exposure assays. Affected loci were characterized and their potential to contribute in virulence of the pathogen was assessed.

Conclusions

The identified mutated loci dacB, dnaK, metN, ywqD, lysC, serC and gbpA are known to encode for virulence factors in other bacteria and are suggested to play a similar role in P. larvae.

Significance and Impact of the Study

The study identified new possible virulence factors for P. larvae genotype ERIC I in an unbiased way. This contributes to the knowledge and understanding of the possible mechanisms used by this pathogen to colonize and kill its host.